The ability to detect nucleic acids or nucleotides at trace levels is required and extremely important in many areas of biotechnology. In the past, tracking and detection of nucleotides was usually performed using radioisotopes. However, these methods employing radioisotopes are generally very laborious, time-consuming, expensive, and require the use of unstable and hazardous radioisotopes leading to problems and handling and disposal of the radioisotope labeled reagents. Therefore, interest has arisen in discovering alternative and safer methods of detection.
One such alternative has been the suggestion that enzyme catalyzed color development be employed However, this proposed methodology has not found general acceptance because of a much lowered sensitivity than methods employing radiolabeled nucleotides In addition, the enzyme catalyzed methodology was found not to have any general improved ease of performance over the radiolabeled nucleotide methods.
Therefore, as another alternative, various methods of detecting nucleic acids or nucleotides based upon fluorescent emissions have been proposed or employed. Perhaps the most widely employed method involves staining with a dye such as ethidium bromide. However, due to background emission from unbound dye, the detection limits cannot approach those in autoradiography. It has been proposed that elimination of the background problem due to free dye can be achieved by covalent modification of nucleic acid with a fluorescent tag followed by separation of unreacted label. With appropriate choice of fluorophore and optimization of the optical train, sensitivities approaching or matching those of radioisotopic detection are considered to be possible Several research groups have employed this approach to detect DNA fragments in polyacrylamide gels. A drawback of this approach is that the gel is a source of significant scattering and background fluorescence.
An alternative detection scheme which is theoretically more sensitive than autoradiography is time-resolved fluorimetry. According to this method, a chelated lanthanide metal with a long radiative lifetime is attached to the molecule of interest. Pulsed excitation combined with a gated detection system allows for effective discrimination against short-lived background emission Syvanen et al., Nucleic Acids Research, 14, 1017-1028 (1986) have demonstrated the utility of this approach for quantifying DNA hybrids via an europium-labeled antibody. In addition, biotinylated DNA was measured in microtiter wells using Eu-labeled strepavidin as reported by P. Dahlen, Anal. Biochem , 164, 78-83 (1982). However, a disadvantage of these types of assays is that the label must be washed from the probe and its fluorescence developed in an enhancement solution. In addition, it has been difficult to provide sufficiently stable labeled molecules to provide for acceptable detection thereof. Moreover, in gel electrophoresis systems the labeled molecules have generally not provided sufficient stability on dilution or when subjected to the elevated temperatures of the gel electrophoresis to enable acceptable detection of the labeled molecules. A further drawback has been the fact that the fluorescence produced has only been in the nanosecond (ns) range, a generally unacceptably short period for adequate detection of the labeled molecules and for discrimination from background fluorescence.
Thus, a need has clearly arisen for fluorescent labeled nucleotides that can be employed in gel electrophoresis systems to provide long lived fluorescence to avoid background fluorescence by use of an intermittent excitation source and a timed coupled measurement of fluorescence. A still further need is to provide for such fluorescent labeled nucleotides for use in detecting nucleotides by time-resolved fluorimetric determination of such labeled nucleotides separated in a gel electrophoresis system in which the labeled nucleotide remains stable and detection limits are significantly improved in comparison to covalent labels with fluorescent lifetimes in the nanosecond range or in comparison to such system employing stains such as ethidium bromide A further need is to provide such a detection method in which the fluorescent labeled nucleotide remains stable and fluorescent upon dilution in the gel system and in an electric field at an elevated temperature of about 60.degree. C. A still further need is to provide such a method for such time-resolved fluorimetric detection of labeled nucleotides in gel electrophoresis systems in which no enhancement solution is required for detection and thereby permitting on-line detection of the fluorescent labeled nucleotides.